Create new candidate hybrids. Planning is in progress for new hybrids to be created during next quarter (Spring 2022), based on parental combinations yielding the best progeny in previous trials. Hybrids from previous crosses were selected and propagated this quarter. New seed source trees for advanced selections were prepared for field planting in Spring 2022.Propagate and plant new field trials. One new Stage 1 field trial with Valencia and one new Stage 2 field trial with Valencia were field planted this quarter. Trees for a Stage 2 Hamlin trial with selected released rootstocks and the best of the next generation hybrids are being grown in the greenhouse in preparation for field planting in Spring 2022. Nursery trees for one new Valencia Stage 1 trial with 60 new rootstocks are being prepared for field planting in 2022. Budwood increase trees were grown in preparation for budding trees for new rootstock trials. Trees with Valencia scion and HLB-tolerant interstocks were propagated as a preliminary analysis of interstock feasibility.Collect data from field trials. Measurements of fruit crop and fruit quality data were collected on 6 rootstock trials with Hamlin and other early season scions. The USDA researcher assisting with the analysis of fruit quality from USDA rootstock trials retired this quarter, so responsibility for this aspect of the work shifted entirely onto the Bowman program. Because of the Bowman program emphasis on rootstock trials, it is anticipated that this change will allow for an increase in the pace of fruit quality analysis associated with the rootstock trials. Evaluate effectiveness for seed propagation of new rootstocks and develop seed sources. Studies continued to evaluate seed propagation for promising SuperSour hybrid rootstocks, using morphological and SSR analyses of seedling progeny for trueness-to-type. Additional effort was initiated on this subject as a graduate student project. As the best rootstocks are identified through field trials, seed sources are established. Cooperative work continues to compare field performance of rootstocks propagated by seed, cuttings, and tissue culture. Evidence indicates that performance of rootstocks is primarily determined by rootstock cultivar, with only small influences by propagation method.Field trial results for grower access. The USDA rootstock trials produce large amounts of information that is useful to help growers make informed decisions about rootstock choice for new plantings. A manuscript is in preparation that has a detailed comparison of field performance for Valencia on 50 SuperSour rootstocks and other commercial rootstocks, and selected excerpts from this were prepared for presentation at the 2022 Florida Citrus Show and the January 2022 meeting of the Florida Citrus Research Foundation. During this quarter, updated trial summaries were prepared for uploading to the USDA citrus rootstock program website https://www.citrusrootstocks.org/, and information was provided to individual growers and groups, as requested.Release of superior new rootstocks for commercial use. Release of new USDA rootstocks is based on robust data from multiple trees in replicated field trials over multiple years. Assessments of trees in trials includes information on tree survival and health, canopy size, fruit yield and fruit quality, and observations on tolerance of disease and other biotic and abiotic threats. Several of the 350 advanced Supersour rootstock hybrids in field trials are exhibiting outstanding performance in comparison with commercial standard rootstocks. Performance data continues to be collected and is being used to critically compare the new hybrids with each other and existing rootstocks. It is anticipated that 2-3 of the most outstanding of the new SuperSour hybrids will be officially released in 2022-23.
This project addressed CPDC priority 3A. Collect standardized data on existing field trials to evaluate citrus scion and rootstock response to HLB. The purpose of the project was to conduct evaluations of rootstocks in existing replicated field trials regarding their horticultural performance and ability to mitigate HLB-induced decline. The cooperative trials were planted in 2015 and contain more than 20,000 Valencia and Hamlin trees on more than 30 different replicated rootstock cultivars and other non-replicated ones. Trials were under commercial management (Lykes Bros.) and planted near Basinger (Highlands County) and Lake Wales (Polk County). For annual evaluations, only replicated rootstocks were included using 6 replications. The complete set of rootstocks was included in year 3 evaluations. The rootstocks included UF/IFAS (CREC) released and experimental selections, California selections (available in FL), and selections from Spain. The Spanish selections are proprietary and not available for use in the USA currently but may be available soon. The UF selections are tetraploid cultivars, meaning that they were created by somatic hybridization or from crosses at the tetraploid level. All other rootstocks were diploids and created by sexual hybridization.All replicated rootstocks were evaluated annually using the methods established in the Denver protocol and outlined in the proposal (also see appendix). Data collected include tree size (height, canopy volume, trunk diameter), fruit yield, fruit quality (total soluble sugars, acid, brix/acid ratio, etc.), leaf nutrients, canopy health (canopy color, canopy density, foliar HLB disease symptoms), and wind resistance/tree leaning (after hurricane Irma). A comprehensive data was assembled for each scion/rootstock combination and trial location and is attached to this report as 4 Excel files: 1) Albrecht_18-029C_VAL_Replicated_Yrs1-3.xlsx; 2) Albrecht_18-029C_VAL_All_Yr3.xlsx; 3) Albrecht_18-029C_HAM_Replicated_Yrs1-3.xlsx; 4) Albrecht_18-029C_HAM_All_Yr3.xlsx.
This project addressed CPDC priority 3A. Collect standardized data on existing field trials to evaluate citrus scion and rootstock response to HLB. The purpose of the project was to conduct evaluations of rootstocks in existing replicated field trials regarding their horticultural performance and ability to mitigate HLB-induced decline. The cooperative trials were planted in 2015 and contain more than 20,000 Valencia and Hamlin trees on more than 30 different replicated rootstock cultivars and other non-replicated ones. Trials were under commercial management (Lykes Bros.) and planted near Basinger (Highlands County) and Lake Wales (Polk County). For annual evaluations, only replicated rootstocks were included using 6 replications. The complete set of rootstocks was included in year 3 evaluations. The rootstocks included UF/IFAS (CREC) released and experimental selections, California selections (available in FL), and selections from Spain. The Spanish selections are proprietary and not available for use in the USA currently but may be available soon. The UF selections are tetraploid cultivars, meaning that they were created by somatic hybridization or from crosses at the tetraploid level. All other rootstocks were diploids and created by sexual hybridization.All replicated rootstocks were evaluated annually using the methods established in the Denver protocol and outlined in the proposal (also see appendix). Data collected include tree size (height, canopy volume, trunk diameter), fruit yield, fruit quality (total soluble sugars, acid, brix/acid ratio, etc.), leaf nutrients, canopy health (canopy color, canopy density, foliar HLB disease symptoms), and wind resistance/tree leaning (after hurricane Irma). A comprehensive data was assembled for each scion/rootstock combination and trial location and is attached to this report as 4 Excel files: 1) Albrecht_18-029C_VAL_Replicated_Yrs1-3.xlsx; 2) Albrecht_18-029C_VAL_All_Yr3.xlsx; 3) Albrecht_18-029C_HAM_Replicated_Yrs1-3.xlsx; 4) Albrecht_18-029C_HAM_All_Yr3.xlsx.
1. Please state project objectives and what work was done this quarter to address them: This report covers the period of September 1, 2021 – November 30, 2021. The objective of this project is to test transgenic ‘Ducan’ grapefruit trees expressing an anti-HLB antibody fused to the FT (Flowering Locus T) protein (FT-scFv protein). We have inoculation tests completed using graft transmission and Asian citrus psyllid (ACP) transmission of hte pathogen (Candidatus Liberibacter asiaticus, or CLas), and a natural infection trial in a grove at USHRL. Graduate student Chad Vosburg finalized his data analysis of the field, psyllid, and grafting mediated CLas infections. His committee approved the final version of his thesis, and Chad is set to graduate in December, 2021. During the reporting period, he revised and finalized his M.S. thesis document as a draft of a publication of the methods and infection results. 2. Please state what work is anticipated for next quarter:In the next quarter, graduate student Chad Vosberg will graduate (December, 2021). The PI will start to revise the draft documents created by Chad for submission to a peer-reviewed journal for publication. 3. Please state budget status (underspend or overspend, and why):This project is underspend partially due to support by the Plant Pathology and Environmental Microbiology Department for Chad’s stipend and tuition. In the next several months, the PI will draw part of his salary support from the grant because he is now the primary person revising the manuscript for submission to a peer-reviewed journal for publication in the next quarter.
The project has five objectives:
(1) Remove the flowering-promoting CTV and the HLB bacterial pathogen in the transgenic plants
(2) Graft CTV- and HLB-free buds onto rootstocks
(3) Generate a large number of vigorous and healthy citrus trees
(4) Plant the citrus trees in the site secured for testing transgenic citrus for HLB responses
(5) Collect the field trial data
Four of the proposed objectives have been accomplished. Objective (5) is delayed due to the Covid-19 pandemic. Activites conducted for each of the objectives are described below.
(1) We previously generated three HLB-tolerent transgenic lines, ‘Hamlin’ 13-3, 13-29, and Duncan ’57-28′. The transgenic plants carried both the HLB-causing bacterium CLas and the flowering-promoting CTV vector. To remove CTV and CLas, the plants were treated in a growth chamber with alternating temperatures of 25 and 42 degree C every 4 hours for a total of 60 to 90 days. New shoots formed on the treated plants were tested for the presence of CLas and CTV by quantitative PCR (qPCR) with specific primers. The alternating temperature treatment is known to be highly efficient for removing CTV. We found that it is also effective for eliminating CLas. The new shoots obtained were free of both CTV and CLas.
(2) & (3) Budwoods from the new shoots were grafted onto ‘Swingle’ rootstocks. A total of 83 transgenic plants were produced using CTV- and CLas-free budwoods of the above mentioned transgenic lines (28 for 13-3, 31 for 13-29, and 28 for 57-28). Moreover, 13 plants propagated from a new NPR1 transgenic line generated through mature tissue transformation, 17 plants from an EDS5 transgenic line, and 7 plants from an ELP3 transgenic line were produced. All these transgenic lines showed HLB tolerance in the greenhouse. In addition, a total of 27 transgenic rootstock plants were produced. These transgenic plants include eight transgenic ‘Carrizo’ lines that express three different disease resistance genes. The transgenic rootstocks were replicated and grafted with ‘Valencia’. All plants were grown and maintained in the greenhouse for two to three years.
(4) The transgenic plants were planted in The Picos Farm in 2019 and 2021 (no plants were planted in 2020 due to Covid-19). A total of 69, 98, and 27 plants were planted on May 9, 2019, May 20, 2021, and October 8, 2021.
(5) This objective has not been finished. The transgenic plants transplanted on May 9, 2019 and May 20, 2021 were examed. The plants grow well in the field and one plant from the 2019 planting has shown HLB symptoms. Field trial data will be collected in the coming years.
Besides the proposed objectives, we continue working on development of techniques that are able to produce consumer friendly citrus products. These techniques include CTV-delivered gene silencing, transgene-free CRISPR, and cisgenesis or intragenesis (cis/intragenesis). Our CTV and CRISPR projects are supported by USDA. The cis/intragenesis project is partially supported with the CRDF funds. We have previously created an intragenic vector. Unfortunately, the efficiency of the vector is extremely low. Our goal is to develop a strategy to significantly improve the efficiency. We are using the citrus 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) mutant (called EPSPS TIPS) that provides tolerance to glyphosate as a selective marker to increase the intragenic vector efficiency. A strong citrus promoter is needed to drive the EPSPS TIPS gene, we are building a system to identify such citrus promoters.
Once the efficiency of the intragenic vector is improved, it can be used to either silence or overexpress a target gene. We are using CTV-delivered gene silencing to identify targets for silencing, which is supported by USDA. We are also screen for genes for overexpression through cis/intragenesis. We recently discovered two nicotinamide adenine dinucleotide (NAD)-binding receptors in the model plant Arabidopsis, which when overexpressed, increase resistance to bacterial pathogens. With the support of the CRDF funds, we generated transgenic citrus expressing the Arabidopsis NAD receptors. The transgenic citrus plants were inoculated with CLas-infected psyllids and are maintained in the greenhouse for HLB symptom development. We are grafting the transgenic scions onto sweet orange rootstocks for easier detection of HLB resistance or tolerance. Furthermore, the citrus genome encodes several putative NAD-binding receptors. NAD-binding activities of two of the putative receptors were tested. These citrus receptors will be overexpressed using the intragenic vector to create HLB tolerance.
In summary, we have accomplished four of the proposed five objective (~70% of the proposed work) and will finsih field data collection in the coming years.
ObjectiveThis project had only one objective which was to support the operation of the facility that would provide service for production of transgenic citrus plants to researchers working towards solution against huanglongbing (HLB) and citrus canker. The Citrus Transformation Facility (CTF) has been a part of the multipronged approach to fight citrus diseases by producing genetically transformed citrus plants that were used to gain important knowledge about possible introduction of tolerance/resistance traits. This knowledge represented a foundation for understanding of effects that introduced gene(s) could have on the growth of transgenic plants and on their ability to sustain and survive diseases. Within the scientific community working in this field there are laboratories without transformation capabilities, and for those research groups CTF was the place where the ideas from projects came to life because of the production of transgenic plants. Major accomplishments per objectiveThe major accomplishment of this project is that it has facilitated the existence of the CTF throughout the last three years. The CTF has undergone the transition into the Educational Business Activity (EBA) unit within the University of Florida that overhauled the way it provides service and operates as business. This transition together with the wide-ranging effects of COVID-19 pandemic have negatively affected operation of the CTF and funding that came from this project together with the funds provided by the University of Florida have kept the CTF open. Within the last three years, the CTF has produced 388 transgenic plants (Table 1). Number of plants produced in 2019 was 246, in 2020 it was 108, and in 2021 it was 36. Without exception, all the plants we created were associated with the research directed towards finding a solution to HLB and citrus canker diseases. Some of our transgenic plants including some Duncan grapefruits and all Pomelos exhibited increased resistance to citrus canker. The other plants we produced have the potential to either be tolerant or resistant to HLB, or in the case of Indian curry leaf plants (Murraya koeinigii), they produce chemicals that can kill Asian Citrus psyllids. Besides Duncan grapefruits and Pomelos, we also produced transgenic Mexican lime, Valencia sweet orange, Kumquats, Pineapple sweet oranges, and Indian curry leaf plants. All of these plants stayed in the state of Florida and were used in tests to determine the efficiency of introduced transgenes on desired traits.Table 1. The plants produced by CTF in the three-year period (2019-2021).Cultivar Number of plants produced Duncan grapefruit 257 Mexican lime 32 Valencia sweet orange 51 Pomelo 22 Kumquat 4 Pineapple sweet orange 10 M. koenigii 12 Total 388 The number of orders placed at the CTF was 57. In 2019, there were 25 orders, in 2020 there were seven, and in 2021 there were 25 orders. The work done on these orders was performed within 266 experiments that included 358 co-incubations of seedling explants with appropriate bacterial strains. Altogether, 314,000 explants were processed in these experiments. Approximately, 170,000 explants were inspected under the microscope equipped with the blue light source for the presence of GFP-fluorescing shoots and buds. Close to 3,000 PCR reactions were done in search of shoots either carrying or expressing a transgene of interest. Also, 1,750 GUS histochemical assays were performed to confirm the transgenic nature of shoot and plants. In average, the transformation rate was about 1% and that number included both chimeric and fully transformed shoots. The percentage of shoots that were successfully grafted was low in the range of 50%.With the help of staff from CREC’s Business office, during the year 2021 I was able to organize the system of payments for the work performed by the CTF as an EBA and get first payments processed.
Fourth quarter 2021- 100% done Project rationale and focus: The driving force for this three-year project is the need to evaluate citrus germplasm for resistance to HLB, including germplasm transformed to produce proteins that might mitigate HLB, which requires citrus be inoculated with CLas. Citrus can be bud-inoculated, but since the disease is naturally spread by the Asian citrus psyllid, the use of psyllids for inoculations more closely resembles “natural infection”, while bud-inoculations might overwhelm some defense responses.CRDF funds supported high-throughput inoculations to evaluate HLB resistance in citrus germplasm developed by Drs. Ed Stover and Kim Bowman for the last 3 years. The funds cover the costs associated with establishing and maintaining colonies of infected psyllids; equipment such as insect cages; PCR supplies for assays on psyllid and plant samples from infected colonies; and two GS-7 USDA technicians. A career base-funded USDA technician also assigned ~30% of her time to the program in order to maintain colonies (including watering, setting up new cages, terminating old cages, cleaning growth chambers and cages). USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations. This quarter: Colonies of CLas infected psyllids supplied a total of 3,770 ACPs used for (1) transgenic events evaluation, (2) applied research for CLas control in citrus performed by USDA and University researchers; and (3) monitoring the colony quality by qPCR.The Stover lab conducted detached leaf assays (DLAs) challenging transgenic citrus with CLas inoculated by infected ACP in the lab, which is used to identify best performing transgenic events (transgenics varying by position of transgene insertion etc.) expressing antimicrobial peptides and defensive proteins targeting CLas, as well as natural insecticide peptides to control ACP. Five detached leaf assays experiments, involving individual 228 leaves, were inoculated using 2,280 CLas infected ACPs in this quarter. Transgenic material tested in DLAs were Carrizo plants expressing ONYX peptide and chimeric AMP TS, both under SCAmp-P3 phloem specific promoter. A total of 38 independent events were tested alongside WT controls. The leaves (midribs) and ACPs are being processed and submitted to qPCR for CLas titer after each DLA to better understand the effect of the transgenic peptide in bacteria control and transmission. These trials have being very useful in terms of providing information that allow to select the best transgenic events (ones causing high ACP mortality and/or low CLas transmission to plant) for propagation and further evaluation at greenhouse environment. We continue to see substantial ACP mortality from feeding on CLas-killing transgenic leaves, with some ONYX events killing around 80% of the psyllids with reproducible results. Research involving evaluation of the microbiome of ACPs fed on transgenic causing high insect mortality was conducted this quarter using 440 ACPs fed in a set of 44 transgenic leaves. A research paper has been prepared (Rapid in vivo screening for huanglongbing resistance in genetically modified citrus by detached leaf assay- J.Krystel, M. Grando, Q. Shi, E. Cochrane, E. Stover) in order to report important modifications implemented into the DLAs using Clas+ ACPs to evaluate transgenic plants and investigate the mode of actions of peptide in controlling the psyllids. In addition, 1,080 CLas+ ACP were provided to researcher collaborators:780 for Florida International University, for Jessica Dominguez, a Ph.D. student, who is developing a thesis in alternative compounds to control CLas bacteria and 300 ACP were furnished to Dr. Randy Niedz (USDA Fort Pierce) for activities in a HLB NIFA project. Periodic colony checks were conducted by PCR to maintain CLas positive colonies. This quarter 410 ACPs were used for Clas detection by qPCR to monitor colony quality. Also, six new colony cages were set up this quarter to renew and support the demand of the hot ACPs. For that 24 HLB positive plants were infested with 2.500 ACPs. Previous quarter: United States Department of Agriculture scientists Kim Bowman, Ed Stover, Michelle Heck, Randy Niedz, and researcher collaborators have all run experiments totaling 6,840 ACPs. Samples have all been collected on-time from ongoing experiments. ACP mortality were computed and statistically analyzed. Surviving ACPs and leaf midribs were collected and processed for future qPCR analysis to access the bacteria CLas titer).
1. Please state project objectives and what work was done this quarter to address them: The objectives of this project were to produce disease resistant, commercially & agronomically acceptable, mature citrus transgenics, intragenics, & GMO/non-GMO edited trees using Agrobacterium as a service for research & commercialization. The research focus of this project was to improve transformation efficiencies, so that the mature citrus protocols become more productive, decrease prices for scientists, & contribute more to financial self-sufficiency of our lab. A biolistic transformation protocol was also developed to overexpress all-citrus intragenic sequences using a citrus gene for selection, which is useful for food crops. A final comprehensive report will be submitted in the near future to CRDF. The efficiency of the mature citrus Agrobacterium transformation protocol was increased significantly, primarily due to the introduction of new cultivars developed by the Plant Improvement Team & determining which ones were not recalcitrant to Agrobacterium. The mature citrus Agrobacterium protocol was originally developed for Pineapple sweet orange but Pineapple is not commercially important to the Florida juice industry. Instead we have found several new sweet orange cultivars that are readily transformed with Agrobacterium. Currently we are focusing on producing non-GMO edited mature citrus & have seen some encouraging results. In addition, we continue to produce all-citrus intragenics using biolistic & Agrobacterium transformation. The plants produced using these technologies might not be as strictly regulated compared to plants produced by transformation with genes from foreign organisms. The USDA APHIS, EPA & FDA will work with each scientist on a case by case basis to deregulate superior intragenic trees. Intragenic trees can likely be provided to the growers relatively rapidly. During the last quarter, ~82 transgenics were produced using Agrobacterium transformation of mature rootstocks & ~87 Cas9 trangenic edited scions were produced for another scientist. We have two new potential projects involving scientists from other states & countries. A manuscript describing a protocol for a new liquid selection system in mature rootstock was published, which showed significantly higher Agrobacterium transformation efficiency in liquid than on solid medium. In addition, Agrobacterium transformation efficiency also increased in mature scions using liquid selection & another manuscript is in preparation. Currently, a manuscript is in review about the new citrus, intragenic selectable marker. It is the responsibility of the scientists to field test their trees & to my knowledge, at least ~ two to three projects are being field tested in the Picos Farm during this last granting cycle. The lab transitioned to an Educational Business Account for earning salaries & operating funds, however it is difficult to earn enough money for salaries by selling plants. Prices are being increased again in 2022 since we will no longer receive CRDF funding.
Objective 1, Mthionin Constructs: Assessment of the Mthionin transgenic lines is ongoing. As the most proven of our transgenics, we continue to use them as a reference in detached leaf assays, as well as studying them in established greenhouse and field trials. The first MThionin field trial (45 plants, WT or transgenic Carrizo with rough lemon scions) has shown transgenics maintaining higher average CLas CT values (2.5 CT higher @ 18 months), but with a high degree of variability. The larger second MThionin planting (205 total grafted plants of transgenic Hamlin scions, transgenic Carrizo rootstock, or WT/WT controls) is producing encouraging results; with the transgenic Hamlin on WT Carrizo having statistically better trunk diameter, tree height and canopy volume compared to controls. Leaf samples from the second planting are collected and undergoing CLas quantification. Results from the 2nd generation plantings detailed below also show the included mThionin lines with significantly improved growth and CLas titer numbers compared to WT when grafted to commercial scions. The Mthionin construct has also been extensively transformed into additional varieties; with 10 confirmed transgenic lines of US-942 and 44 putative lines of Valencia and Ray Ruby undergoing expression analysis. Objective 2, Citrus Chimera Constructs: Detached leaf assays, with CLas+ ACP feeding, have been conducted to screen citrus lines expressing chimera constructs TPK, PKT, CT-CII, TBL, BLT, LBP/’74’, ’73’, and ‘188’ (as well as scFv-InvA, scFv-TolC, Topaz and Onyx). Testing of all 35s driven Carrizo lines is complete and the analysis of phloem specific and scion-types is well underway. This work has already identified numerous lines with significant effects including increased ACP mortality (up to 95% from TBL, 96% from PKT, 60% from BLT and 70% from TPK) and decreased transmission of CLas into the leaf. Analysis of the ACP endosymbionts show a reduction in titer that may indicate a mode of action for ACP mortality (outlined in Objective 4). The best performing of these lines have been moved forward into greenhouse trials as described below. Initial ACP-inoculated greenhouse trials on 8 lines of citrus Thionin-LBP chimeras (’73’, and ’74’) showed a statistically significant reduction (13x) in CLas titer for ’74’ transgenics vs WT. However, many plants of both treatments remained CLas negative due to low inoculation efficiency. Two follow on greenhouse studies are underway. The first is an improved ACP-inoculated trial with the best performing `74′ and `188′ lines. The second is a larger graft-inoculated study directly comparing the best performing 3rd generation chimeras (TPK and TBL) with the earlier 1st (Mthionin) and 2nd (`74′ and `188′) generation lines. For this, a total of 420 grafted plants (all on WT Carrizo rootstock for uniformity) were made and grafted with CLas+ RL for uniform transmission. In the earlier 6 and 9 month samplings, plants were beginning to test CLas positive and 12 month assessments are underway. An additional two rounds of rooted cuttings (totaling >1600 replicates) have been made from those same lines for paired ACP-inoculated greenhouse and field trials. ACP-inoculations will be done in free flying cages for a 2 month availability window, a protocol developed in a parallel project that has yielded up to a 100% infection rates. Both studies will begin as soon as the plants are large enough. The best performing PKT and LBT lines have also been replicated (>200 plants) for following the same greenhouse to field study pipeline, with graft inoculations as soon as the plants are of sufficient size. Field trials of 2nd generation chimeras (`74′, and `188′) with included MThionin plants are ongoing; with 165 plants (WT Hamlin and Ray Ruby on transgenic Carrizo) and 70 plants (WT Valencia on transgenic Carrizo) moved to the field in August 2020 and May 2021 respectively. At the 1 year assessment, Hamlin plants with transgenic Carrizo rootstocks showed noticeably reduced CLas titer (in scion midribs) when compared to those on conventional rootstocks. WT Hamlin on `74′ Carrizo had a 10x reduction in CLas titer, WT Hamlin on MThionin a 15x reduction and WT Hamlin on `188′ a 144x reduction. Plants grafted on MThionin-Carrizo also showed better trunk diameter, tree height and canopy volume compared to WT/WT grafts. Because of the high ACP mortality seen in detached leaf assays, field plantings of all chimera are also being surveyed to determine the rates of successful ACP colonization on new flush. An additional planting of transgenic scions (Hamlin) on WT rootstocks is also being prepared to complement these plantings. Eighteen new transformations, totaling over 6200 explants, have been completed to generate sufficient events of Valencia, Ray Ruby, US-942, and Hamlin lines expressing `74′, `188′, TBL, TPK and other advanced chimera constructs. From this effort, over 325 new lines from 74-Valencia, 74-Ray Ruby, 74-US-942, 74-Hamlin, 188-Ray Ruby, 188-Valencia, 188-US-942, TBL-US-942, TBL-Hamlin, TBL-Ray Ruby, TPK-Ray Ruby, TPK-US-942 and TPK-Hamlin are now in soil. Transgene expression analysis has confirmed the first 29 of these lines as positive with the remainder still being tested. In addition to the use of the Mthionin and its chimeric variants, new strategies have been implemented in our laboratory to develop HLB resistant citrus. These efforts include the expression of insecticidal peptides (to control ACP) and down-regulation of DMR6 genes (to enhance disease resistance). 54 independent transgenic lines of Carrizo, Hamlin and Ray Ruby expressing the insecticide peptide Topaz (a code name to protect IP), under constitutive and phloem specific (SCAmpP-3) promoters were evaluated by detached leaf assay. From these, 12 lines (4 event of each genotype) showed significant ACP mortality and were selected to move up in the screening pipeline for HLB/ACP tolerance. Also, 27 transgenic Carrizo and Hamlin lines highly expressing Onyx (a code name to protect IP), a peptide with both antimicrobial and insecticide activity, were evaluate by DLA. The 5 Onyx-Carrizo lines showing high ability to kill ACP (to 83% mortality) were selected for further evaluation. Strongly performing lines were replicated as rooted cuttings (250 Onyx and 189 Topaz plants) that will soon enter greenhouse trials. The available Onyx transgenic material is being further expanded through production of additional constitutive (13 Valencia and 6 Ray Ruby) and phloem specific lines (25 Carrizo, 5 Hamlin, 8 Valencia, and 13 Ray Ruby). Down regulated DMR6 Carrizo, either by stable expression of specific hairpin RNA (for RNA interference) or by Cas9-sgRNA genome editing were generated, cloned, and are being assessed. Since DMR6 is a broad immune suppressor, down-regulated plants could be expected to have heightened immune response. To test this, they were first evaluated for potential Canker resistance which is both a quicker assay and a desirable trait in its own right. After Xanthomonas challenge on detached leaves, both hairpin and gene edited DMR6 lines showed reduced bacterial titers, statistically significant reductions in Canker symptoms and higher expression of some down-stream defense genes compared to WT controls. Several transgenic lines developed no disease symptoms whatsoever. A planting of 190 trees (including WT controls) from the best performing genome edited line (40 trees) and hairpin knockdown lines (150 trees) is being prepared; the plants have been replicated as rooted cuttings and will go into the field once regulatory requirements are met. An amendment to the current BRS permit has been submitted for their addition and is currently under review. As an effort to accelerate development of non-transgenic HLB resistant plants using gene editing, we transformed early flower transgenic plants (carrying FT-Mcherry or FT-scFv gene) with the DMR6 targeting CRISPR construct. Cotyledons from FT-Mcherry line #3 seeds were transformed and 51 putative positive plants are now growing in soil. These plants are already showing strong early flowering phenotypes, but must still be confirmed for presence of the CRISPR transgene. Seeds from 4 more FT-Mcherry lines are now being germinated for additional transformations. 50 additional plants from FT-scFv/CRISPR stacking transformations are also in soil awaiting confirmation. The early flowering trait will greatly decrease the time needed to produce an edited but non-transgenic offspring. A set of 30 confirmed positive plants from this gene stacking effort will be evaluated. Objective 3, ScFv Constructs: ACP inoculated greenhouse studies on 5 scFv lines have been completed with transgenics showing significantly reduced CLas titer (up to 250x reduction) and a significantly higher incidence of no CLas rDNA amplification in roots and leaves compared to WT. These lines have been grafted with WT Ray Ruby scions and are undergoing field trials at Picos farm. The one year assessment is now complete and several scFv lines are showing significantly improved growth characteristics, leaf tissue was collected and is awaiting CLas quantification. An additional 129 rooted cuttings are propagated for follow up plantings with grafted Hamlin scions. A second greenhouse trial testing new lines (150 plants from 12 lines) have been bud inoculated with HLB+ RL. A group of 370 plants for a third greenhouse trial has been propagated with the first 54 plants to reach a suitable size ACP-inoculated using the improved protocol. Plant tissue from both second and third (partial) greenhouse trials has been collected and processed; now awaiting qPCR analysis for CLas quantification. Objective 4, Screening Development and Validation: A protocol using a high throughput ACP homogenate assay for selecting lytic peptides for activity against CLas is now in use. A manuscript on the protocol has been published in Plant Methods (DOI: 10.1186/s13007-019-0465-1) to make it available to the HLB research community. Transgenic Nicotiana benthamiana plants expressing His-6 tagged variants of the chimeras TBL, TPK, PKT and LBP have also been generated to produce sufficient protein extracts for use in exogenous applications in both whole plant and detached leaf assays. The detached leaf ACP-feeding assay (DLA) has undergone several small revisions to improve sensitivity and maintain consistent inoculation; adjusting feeding period and ACP numbers. We have also expanded the analysis of ACP bodies to include quantification of other major endosymbionts (Wolbachia, Carsonella and Profftella) to better investigate the activity of peptides causing CLas mortality. To further investigate the impact of transgenic or exogenous chimeral application, a DLA protocol to assess changes in ACP inoculability is also being developed. A manuscript detailing the protocol and findings is being prepared. An array of phloem specific citrus genes has been selected for investigation as potential reference genes to improve detached tissue and plant sampling techniques. Multiple sets of sequence specific qPCR primers for each gene have been synthesized and tested for efficiency. Six varieties of citrus have been propagated for endogene stability testing. A phloem specific endogene would allow normalizing to phloem cells, more accurately evaluating CLas titer relative to Citrus DNA and potential therapeutic effects. The best performing lines of Mthionin, chimeras `74′,`188′, TPK, TBL, DMR6 knockdowns and scFv transgenics have been submitted to Florida Department of Plant Industry for shoot-tip graft cleanup in preparation for future field studies. Hamlin/Mthionin transgenics (3 lines), Carrizo/Mthionin (2 lines), Carrizo/’74’ (1 line), Carrizo/’188′ (1 line) and Hamlin/’74’ (1 line) have been returned certified clean. Objective 5, Transgenic Product Characterization: Experiments are also underway to track the movement and distribution of transgene products using antibodies and affinity tagged protein variants. CLas+ RL have been grafted as scions onto MThionin expressing Carrizo as a platform to test peptide movement and effects across the graft union. Transgenic Carrizo lines expressing His6 and/or Flag tagged variants of chimeric proteins TBL (15 lines), BLT (15 lines), TPK (17 lines), PKT (20 lines), scFv-InvA (34 lines) and scFv-TolC (30 lines) have been generated and confirmed by RT-qPCR. Total protein samples have been extracted from His-tagged transgenic lines and sent to our collaborator for testing.
This project has two objectives: (i) Assess the performance of new grapefruit cultivars with certain rootstocks in the IR district; and (ii) Evaluate the influence of UFR and other recent rootstocks on grapefruit, navel, and mandarin in the IR in comparison to legacy/standard rootstocks. There are four trials: Trial 1) 18 grapefruit cultivars on three rootstocks; Trial 2) 32 rootstocks with `Ray Ruby’ grapefruit as the scion; Trial 3) 31 rootstocks with ‘Glenn F-56-11′ navel orange as the scion; and Trial 4) 31 rootstocks with `UF-950 mandarin as the scion.There are now 4,900 trees in the grove. The final 90 grapefruit trees on UFR-8 rootstock are growing slowly in the nursery and are expected to be planted in February or March 2022. In August 2021, leaf and soil samples were collected from each experimental plot to properly manage fertilizer requirements. Controlled-release, polycoated fertilizer was applied appropriately in early November 2021 based on lab results. All trees were treated biweekly with appropriate agrochemicals to manage canker, Asian citrus psyllid, mites, and citrus leafminers.Tree height, tree width in cardinal directions (E-W/N-S), and trunk diameter were measured on the three middle trees in each experimental plot in October 2021 to quantify canopy volume and tree size. Some scion/rootstock combinations are exhibiting significant differences in canopy volume. In Trial 1, `Pummelette UF-5-1-99-2′ grapefruit on US-942 is 5.6X larger (11.2 m3) than `US 1-83-179’ grapefruit hybrid on sour orange (2.0 m3). In Trial 2, grapefruit on UFR-15 is 2.6X larger (8.9 m3) than on 46×20-04-6 (3.4 m3). In Trial 3, navel orange on US-802 is 1.8X larger (5.3 m3) than on Willits, UFR-16, and UFR-1 (2.9 m3). In Trial 4, mandarin on US-942 is 2.6X larger (6.2 m3) than on 46×20-04-6 (2.4 m3). Many trees are developing vigorous canopies despite HLB symptoms. Results will be presented at the annual Florida Citrus Show in January 2022 in Fort Pierce, FL. Visual HLB symptoms are apparent on approximately 25% of the total tree canopy volume for most of the plots. However, trees look vigorous and maintain a bright green foliage. Leaf samples from Spring flush were collected in September 2021 and sent to Southern Gardens Labs for quantifying CLas titer. The ct values ranged 35-40 (CLas-free >32). Leafminer damage was substantial during the summer months but decreased recently with cooler temperatures. Nevertheless, tree growth has not been significantly adversely affected by these pests due the biweekly application of agrochemicals.Trees are bearing their first fruits, and we expect to collect this information in the first quarter of 2022, which will serve well to establish potential yields and marketability as well as HLB damage. Long-term evaluation is needed to identify the most promising scions and rootstocks to determine their profitability and capability of meeting grower and market needs.The Citrus Horticulture Lab organized the annual drive-through Millennium Grove Field Day on 14 October 2021 to showcase the results to growers and stakeholders. More than 50 attendees coming from local and neighboring counties (St. Lucie, Okeechobee, Polk) toured the grove, received an explanatory handout, and met with personnel involved in the project.
We generated raw sequence data for Valencia orange (S, sensitive), Ruby Red grapefruit (S), Clementine mandarin (S), LB8-9 Sugar Belle® mandarin hybrid (T, tolerant), and Lisbon lemon (T) and preliminary assemblies and analyses were carried out. Because of reduced sequencing costs, we were able to enter additional important genomes into the pipeline beyond those originally proposed, including Carrizo citrange, sour orange, and Shekwasha (an important breeding parent for HLB tolerance); these also have now been sequenced and preliminarily assembled. The transcriptome data for two of our target genomes, produced previously by a commercial vendor on their PacBio platform was found to be inadequate. So, we are in the process of finding another vendor to redo the long-read sequencing on the most recent PacBio platform. Hi-C sequencing of the 7 remaining target genomes is underway; two were completed. These Hi-C results will be combined with the PacBio assemblies using Hi-Rise software, to produce improved chromosome scale assemblies. RNA samples of these 7 additional genomes have been prepared from young and old leaf tissue to generate the transcriptome data required for genome annotation, and further characterization of large-scale structural variations within and among the genomes upon which we are focused. However, we are attempting to increase the types and numbers of tissues sampled to have a fuller representation of the gene content of each genome.Finally, we have collaborated with the USDA Germplasm Repository for Citrus and prepared genomic DNA from more than 120 citrus species, varieties, and relatives for enrichment and sequencing of specifically targeted genes. Sequencing libraries have been prepared and are in que for processing. Results from this effort will provide important insights for the evolution and domestication of select genes that are important for citrus resistance or susceptibility to Huanglongbing and other diseases. The results will also provide template gene sequences for genome editing for Huanglongbing resistance.
1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. Seed from a first group of rootstock crosses was harvested and planted in the calcareous/Phytophthora soil as the first step in the `gauntlet’ screen; parents included several previously selected but unreleased HLB-tolerant rootstocks, as well as some of the UFRs, HLB-tolerant pummelos, and US-897 and US-942. Seed were harvested from a second group of crosses, using LB8-9 Sugar Belle® as a seed parent with pollen from various hybrids of Poncirus trifoliata with citrus accessions, Citrus ichangensis (Ci), different Cleopatra mandarin x Ci hybrids, and a Palestine sweet lime x Ci hybrid; these will be planted next into the `gauntlet’ screen. In collaboration with researchers at IFAPA in Spain, new information has been generated regarding performance of selected UFRs and other unreleased rootstock hybrids from our program in response to drought and flooding, Phytophthora, and salinity. Three new rootstock candidates were entered into the Parent Tree Program, including the first LB8-9 Sugar Belle® x trifoliate orange hybrid selected through the `gauntlet’ pipeline.2. Develop new, HLB-tolerant scion cultivars from sweet orange germplasm, as well as other important fruit types such as grapefruit, mandarins, and acid fruit. We removed previously planted and tested scions from our program, grown in the Trailer Park block, that do not warrant further scrutiny. We planted 60 new scion selections from the program that have gone through the DPI PTP cleanup and certification; these include true oranges and orange-like hybrids, grapefruit and hybrids, mandarins, lemons, pummelos, and acid fruit. All trees are on UFR-5 rootstock. Following extensive phenotyping of a replicated planting of hybrids between Monreal Clementine and an accession of Citrus latipes (perhaps the most HLB-tolerant citrus), we have found at least two hybrids that remain PCR-negative after 6 years under high pressure in the field. They fruited last season and produced large fruit, somewhat resembling sweet orange but with high acidity. Pollen was collected from these and used to make crosses this past spring with two low-acid selections in our breeding program. Several thousand seeds were produced and will be planted soon. We plan to screen the seedlings with markers previously developed for fruit quality, acid content, and potential HLB tolerance. We completed micrografting somaclone seedling-derived populations of early maturing (January), high soluble solids OLL sweet orange clones to UFR-4 rootstock liners, in efforts to generate an even earlier maturing OLL clone. More than 100 individual seedlings successfully grafted to UFR-4 were stepped up to 4×4 citripots for spring planting at St. Helena. Parent Tree Program scion entries included 2 HLB-tolerant Valencia mutants produced by irradiation and selected from a small, replicated trial, and 1 September-maturing mandarin hybrid. Finally, 125 triploid hybrids, including sweet orange-like, mandarins and grapefruit, were prepared for field planting at the CREC. 3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. We have more than 70 5-year-old-trees of `Marathon’ mandarin on sour orange, that set huge crops of fruit this season. Although all trees have HLB, there are few to no obvious disease symptoms in fruit, leaves, or canopy, demonstrating a high degree of tolerance thus far. We have followed closely their performance, and individual trees yielded more than 300 pounds of fruit. A first harvest was made in late September, and another planned for October. We will determine fruit size distribution, and we will follow post-harvest behavior and fruit quality. 4. Conduct studies to unravel host responses to CLas and select targets for genetic manipulations leading to consumer-friendly new scion and rootstock cultivars. Several new genetic constructs have been developed using newly identified citrus specific promoters (phloem and root tissue), and new putative disease resistance genes, or downstream genes. Transgenic plants have been produced with some of these constructs, and additional transformation experiments are underway with several sweet oranges, grapefruit, and rootstocks. Finally, we have completed metabolomic studies, in collaboration with Dr. Y. Wang, to gain insight into the underlying mechanisms of HLB-tolerance and sensitivity; please see doi: 10.3389/fpls.2021.710598 and doi: 10.1021/acs.jafc.1c02875.
The objectives of this project are: 1. Evaluate existing transgenic Carrizo and Swingle AtNPR1 overexpressing rootstocks in the laboratory and greenhouse. 2. Conduct a replicated field trial with the best transgenic rootstocks budded with non-transgenic `Valencia’ and test for GMO gene products in the fruit or juice. 3. Produce additional transgenic rootstock lines and stack other gene(s) responsible for SAR using mature transformation. 4. Evaluate transgene segregation analyses of the rootstock progeny and large-scale propagation of select lines Obj 1: Transgenic rootstocks (n=12) that were budded with non-transgenic scion were side grafted with HLB infected budwood and maintained in the greenhouse. 6 months following infection trees were tested for the presence of HLB and were also evaluated for PR1 gene expression. PR1 is a SAR marker. We observed that all scions (with transgenic rootstock or non-transgenic control) were infected within a year of inoculation. There was no statistical difference between the treatments and control for the first 18 months. In several transgenic rootstock lines, Ct values did not decline at the same rate as controls after 18 months of infection. In several transgenic rootstock – non transgenic scion combinations, there was also an enhanced expression of the PR1 gene, which indicated an active defense mechanism. At the termination of the project, all lines with enhanced PR1 gene expression were alive, albeit infected. 4 of the 12 controls died while 6 more exhibited enhanced HLB symptoms. Only one control could be considered an outlier with mild HLB symptoms at the termination of the project. Obj 2: This objective was in progress at the termination of the project with rootstock lines clonally propagated in the mist bed and ready to be budded with non-transgenic scion. Obj 3: 61 transgenic lines (Carrizo, US942) with different genes stacked with NPR1 were produced by the mature transformation lab. Most of these tested were determined to produce adequate transprotein. At the termination of the project, all lines were being sized up in the greenhouse for clonal propagation. Additionally, several select lines were budded onto standard trifoliate rootstocks for field planting. This was being planned for seed production. Obj 4: This objective – field based under USDA BRS permit could not be initiated due to termination of the project
True sweet oranges: With the passing of Louise Lee, the trial block containing the OLL somaclone seedling population is in jeopardy of being sold for development. Efforts to rescue the most promising clones from these trial blocks continued. Multiple selected OLL and Vernia clones have been propagated under permit from DPI, and will be planted at St. Helena. Potential HLB tolerance/resistance from ‘gauntlet’ rootstock candidates: Rootstock sprouts were recovered from 12 superior gauntlet trees that had the tops cut off to induce sprouting for rootstock recovery. All have been successfully grafted to recover the 12 diverse rootstock genotypes (seed trees of these selection were not available). Following clean-up, these rootstocks will be propagated by cuttings and TC micropropagation for avanced trials. 10 more promising rootstock selections were provided to Agromillora for TC micropropagation initiation. These include 3 more promising gauntlet SugarBelle hybrids (one SugarBelle x trifoliate orange 50-7 hybrid), and hybrids of [salt tolerant HBPummelo x Shekwasha] with trifoliate orange 50-7 and x639. Agromillora has already generated 3500 shoots from the promising S10xS15-12-25 gauntlet rootstock hybrid previously introduced. This collaboration with Agromillora will greatly accelarate the planting of important stage 2 rootstock trials. CREC Trailer Park Trial: 10 trees each of 65 promising new scions available in the Parent Tree Program (selected based on feedback from Fruit Displays and breeder intuition), all on UFR-5 rootstock, were wrapped, planted and mapped. Trees were grown at Southern Citrus Nursery. Overage trees will be planted at 3 other diverse locations. This will provide a valuable resource to our new hire Dr. John Chater regarding advanced trials and commercialization of the best selections.Rootstock candidate identified from Strang/Gapway trial. In November of 2019, we planted approximately 100 HLB+ Valencia trees on selected rootstocks (good trees left over from a CRDF-funded greenhouse nutrition/rootstock study) in the Gapway grove near the CREC, under permit from DPI. Most of the trees were successfully established. This summer we identified one superior tree, that even with a 24 ct value before planting, grew off twice as fast as any of the other trees, and already set a good crop of fruit, and showed no HLB symptoms. We cut this tree to induce rootstock sprouts, and the rootstock genotype has been recovered. SSR marker analysis shows that the rootstock is from x639, but flow cytometry analysis suggests that there has been a deletion, as the tree repeatedly shows less than the typical diploid amount of DNA. Further investigation is underway. St. Helena: Following removal of all under-performing trees, the irrigation system was repaired and the grove prepared for the major resetting operation. Approximately 600 reset trees are ready to plant, including a new population of Vernia somaclone-derived seedlings, rescued promising selected OLL and Vernia somaclone seedling-derived clones from the Orie Lee trials in St. Cloud (mentioned above, permission granted from DPI to plant rescued sweet orange genotypes at this location), new sweet orange/rootstock combinations, and some new early and mid-season orange-like hybrid scion candidates. Planting is expected to begin this quarter. CREC Block 16: a new population of 175 protoclones of EV-1 and EV-2 were planted in efforts to identify a more robust clone of Early Valencia, and possibly a seedless clone. At present, the available EV clones are showing an HLB response similar to Hamlin regarding fruit drop. Field Trial Data Collection, etc.: Tree height data was collected from the Duda, Peace River and CREC Teaching Block trials. Seed fruit from UFR and a few other promising rootstock selections was harvested for seed extraction (seed to be extracted by Southern Citrus Nursery). Fruit was sampled from all scion trial blocks, and promising early-maturing selections were identified and included in the October Fruit Display – data forthcoming. Data analysis and entry onto the Rootstock Data Website: annual updates included: Premier Indian River grapefruit trial, Greene Citrus lemon trial, IMG navel & grapefruit/rootstock trial, Duda Vernia/rootstock trial, St. Helena rootstock Survey trial, and CREC scion/rootstock trial. Trial data from 20 additional trials is being prepared for website updates, including 8 new trials not yet posted. Initiated creation of a database using Microsoft Access with information from all field trials.
The purpose of this project is to optimize the CRISPR technology for citrus genome editing. This study is related to the CRDF RMC-18 Research Priorities 4AB. Objective 1. Expanding the toolbox of citrus genome editing. In this study, we will adapt StCas9, NmCas9, AsCpf1 (from Acidaminococcus), FnCpf1 (from Francisella novicida) and LbCpf1 (from Lachnospiraceae) on genome modification of citrus. Lately, we have shown CRISPR-Cpf1 can be readily used as a powerful tool for citrus genome editing. In our recent study, we employed CRISPR-LbCas12a (LbCpf1), which is derived from Lachnospiraceae bacterium ND2006, to edit a citrus genome for the first time. Our study showed that CRISPR-LbCas12a can readily be used as a powerful tool for citrus genome editing. One manuscript entitled CRISPR-LbCas12a-mediated modification of citrus has been published on Plant Biotechnol J. We are currently further optimizing LbCas12a-crRNA-mediated genome editing to make homologous biallelic mutations. We are also testing AsCpf1 and FnCpf1 for their application in citrus genome editing and generating homologous biallelic mutations. We have successfully generated both homozygous and biallelic mutations in the EBE region of LOB1 gene in pumlo. This work has been submitted for publication. We are in the process of generating homozygous and biallelic lines of other citrus varieties.Recently, we have developed multiplex genome editing toolkits for citrus including a PEG mediated protoplast transformation, a GFP reporter system that allows rapid assessment of the CRISPR constructs, citrus U6 promoters with improved efficacy, tRNA-mediated or Csy4-mediated multiplex genome editing. Using the toolkits, we have successfully conducted genome modification of embryogenic protoplast cells and epicotyl tissues. We have achieved a biallelic mutation rate of 44.4% and a homozygous mutation rate of 11.1%, indicating that the CRISPR-mediated citrus genome editing technology is mature and could be implemented in citrus genetic improvement as a viable approach. In addition, our study lay the foundation for non-transgenic genome editing of citrus. One manuscript entitled Development of multiplex genome editing toolkits for citrus with high efficacy in biallelic and homozygous mutations has been published on Plant Molecular Biology.We have successfully developed base editing tools for citrus genome editing. This method has been succefully used to generate non-transgenic biallelic mutants of sweet orange. Objective 2. Optimization of the CRISPR-Cas mediated genome editing of citrus. In this study, we are testing different promoters including INCURVATA2 promoter, the cell division-specific YAO promoter, and the germ-line-specific SPOROCYTELESS promoter, and ubiquitin promoter in driving the expression of Cas9 and Cpf1 orthologs. To optimize the expression of sgRNA and crRNA, we have identified multiple citrus U6 promoters and two of the citrus U6 promoters showed higher efficacy in driving gene expression in citrus than 35S promoter and Arabidopsis U6 promoter. We have further increased the mutation efficacy to 50%. We have further optimized the CRISPR/Cas9 system. Now, the biallelic mutation rate reaches 89% for Carrizo citrange and 79% for Hamlin sweet orange. We have generated one homozygous line in the promoter region of canker susceptibility genes of Hamlin. We have successfully generated one biallelic mutant of grapefruit that is canker resistant. We also successfully generated multiple biallelic and homozygous mutant lines of sweet orange that are canker resistant. Objective 3. Optimization of the CRISPR technology to generate foreign DNA free genome editing in citrus. We have conducted transient expression of Cas9/sgRNA plasmid and Cas9 protein/sgRNA ribonucleoprotein complex in citrus protoplast. We are also conducting citrus genome editing using Cpf1/crRNA plasmids and ribonucleoprotein complex in citrus protoplast. The plasmid-transformed protoplast has 1.7% editing efficiency, and the RNP-transformed samples have approximately 3.4% efficiency. The genome modified protoplast cells are undergoing regeneration. We aim to increase the efficacy to over 20% and eventually generate non-transgenic genome modified citrus. One patent has been filed on the CRISPR-Cas mediated genome editing of citrus. We have lately optimized the citrus protoplast isolation and manipulation, our data showed that more than 98% of the isolated protoplasts were alive. We regularly obtained a transfection efficiency of approximately 66% or above. ErCas12a has been succes for non-transgenic gene editing of embryogenic Hamlin sweet orange protoplast cells. We are editing 6 putative HLB susceptibility genes for sweet orange. One biallelic mutant line has been generated for ACD2. We have successfully adapted the adenine base editors (ABE) to modify the TATA box in the promoter region of the canker susceptibility gene LOB1 from TATA to CACA in grapefruit and Hamlin sweet orange. Inoculation of the TATA-edited plants with the canker pathogen Xanthomonas citri subsp. Citri (Xcc) demonstrated that the TATA-edited plants were resistant to Xcc. In addition, cytosine base editors (CBE) was successfully used to edit the acetolactate synthase (ALS) gene of Carrizo citrange, a hybrid of Citrus sinensis `Washington’ sweet orange X Poncirus trifoliata. Editing the ALS genes conferred resistance of Carrizo to the herbicide chlorsulfuron. Two ALS-edited Carrizo plants did not show green florescence although the starting construct for transformation contains a GFP expression cassette. We performed PCR amplification for Cas9 gene in the mutant plants and found that Cas9 gene was undetectable in the herbicide resistant citrus plants. This indicates that the ALS edited plants are transgene-free, representing the first transgene-free gene-edited citrus using the CRISPR technology. In summary, we have successfully adapted the base editors for precise citrus gene editing. The CBE base editor has been used to generate transgene-free citrus via transient expression.
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